Recent data indicate that accumulate damage to mitochondrial DNA (mtDNA) may play an important role in cardiac myopathy, stroke , and other age-related pathologies such as neurodegeneration. In fact, it has been proposed that the accumulation of mtDNA damage plays a fundamental role in normal aging. In addition, a human genetic disease (autosomal dominant progressive external ophthalmoplegia, adPEO) has recently been described in which mtDNA instability can lead to premature death. These observations highlight the deleterious health consequences of damage to mtDNA. Yet comparatively little is known about DNA repair of mtDNA in mammalian cells. A series of recent observations have provided new information about this process: (1) A novel mitochondrial-specific DNA ligase has been identified, (2) Mitochondrial protein extracts prepared from a variety of mammalian cells possess potent DNA end-joining activity, (3) The products of this end-joining activity bear a striking resemblance to mutant mitochondrial DNA molecules observed in vivo, and (4) A DNA end- binding activity has been identified in mitochondrial protein extracts. Based on these observations, we have hypothesized that mammalian mitochondrial possess a non-homologous end-joining DNA repair pathway, analogous to that which functions in the nucleus. The experiments described in this proposal are designed to test this hypothesis. Specifically, gene targeting will be used to inactivate the mitochondrial DNA ligase gene, and the mtDNA repair/stability phenotype of 'knockout' cells evaluated. In addition, the mitochondrial DNA end-binding gene will be cloned. Gene targeting will be used to create mutant cell lines in which this gene has been inactivated, and the mtDNA repair and stability phenotype of these cells will be determined. Based on preliminary results presented in this proposal, it is reasonable to predict that the knockout cell lines described above could possess a mtDNA mutator phenotype. If this provides to be the case, future studies could be devoted to the creation of similar gene inactivations in mice, thereby permitting a test of the hypothesis that accumulated mtDNA damage influences aging.